Project Summary Copper homeostasis plays an essential role in a normal development and function of the cardiac, vascular, and central nervous systems, cholesterol metabolism, and immune response. The main role in the maintenance of copper balance in human cells belongs to the copper-transporting ATPases (Cu- ATPases) ATP7A and ATP7B. We have recently discovered the previously unknown roles for the intestinal ATP7B in maintaining colon morphology and Cu distribution. To characterize these new functions of ATP7B in a physiologically relevant context we have established a novel experimental system based on primary three-dimensional enteroids and defined three main experimental goals. The experiments proposed under Specific Aim 1 will determine the role of ATP7B in regulation of the dietary copper uptake and activation of copper-dependent enzymes in the duodenum. Specific Aim 2 will characterize the novel inter-vesicular route of ATP7B trafficking and elucidate the mechanisms involved in ATP7B movement between the vesicles. Studies under Specific Aim 3 will establish how inactivation of ATP7B causes pathologic changes in the colon. The proposed experiments will develop the fundamental concepts related to physiologic regulation of a mammalian copper homeostasis. The results are expected to facilitate the development of treatments for the known and emerging disorders of copper misbalance.